Method of Lysis of Erythrocytes

ABSTRACT

A multifunctional reagent for erythrocytes containing an amount sufficient to produce the lysis of erythrocytes or the sphering of erythrocytes in such a way that they can be detected by a cytometer or an automatic counting device, of a carbamate or of an agent inducing the formation by the erythrocytes, from carbonate and from a nitrogenated heterocycle or ammonium ions, of a carbamate combined with the absorption of CO 2  by the erythrocytes, process for lysing or sphering erythrocytes and preparation process for leucocytes.

This application is a divisional of patent application Ser. No.09/951,903, filed Sep. 4, 2001, which claims priority under 35 U.S.C.§119(a)-(d) of French Patent Application No. 0011746, filed Sep. 14,2000. All parent applications are incorporated herein by reference intheir entirety.

The present invention relates to new reagents and methods for thetreatment, and in particular the lysis of erythrocytes.

Red corpuscles are approximately one thousand times more numerous thanleucocytes, they form a barrier to the analysis of the leucocyticfraction of the blood. Treatment of blood with a lysis reagent emptiesthe red blood cells of their contents and makes it possible to isolatethese cells from the analysis. An ideal lysis process comprises completelysis of all the erythrocytic elements, without the slightest effect onthe morphology and the viability of the leucocytes.

Hypotonic lyses are known among the numerous lysis reagents and methods.But a deformation of the leucocytes is observed.

Detergents are also known, but these produce a deterioration in themembrane of the leucocytes which are therefore counted by default.

Small neutral, generally lipophilic molecules are also used such asalcohols or aldehydes but these have the drawback of being toxic for theleucocytes.

In fact, reagents containing amines have performance values closer to anideal lysis process. The lysis reagents containing amines include theammonium chloride reagent, the use of which is very widespread and thereagent based on nitrogenated heterocycles, as described in FR-A-2 778413. Lysis reagents with ammonium chloride contain 155 mM NH₄Cl, 10 mMKHCO₃ and can sometimes also contain 0.1 mM EDTA.

However, as described in FR-A-2 778 413, as the ammonium chloride lysisreagent has a rapid and complete lysis effect, it has a toxic effect onthe leucocytes. The problem of the non-specific toxicity of ammoniumchloride is probably linked to the presence of an ammonia concentrationof approximately 1 mM in the lysis mixture. The use of stronger bases,such as pyrrolidine and piperidine with a free base concentration ofapproximately 10 μM, resolves the problem of non-specific toxicity, butgives a slower lysis reaction, this which is a drawback for its use inroutine laboratories.

It would therefore be desirable to have available new reagents and lysismethods for erythrocytes with a more rapid and more complete action.

After much research the Applicant has surprisingly discovered that lysisreagents, and in particular reagents containing amines in the presenceof a carbamate and/or of a catalyst of the reactionCO₂+H₂O

H₂CO₃or/and of the reactioncarbonate

carbamate+H₂Owhich are the two reactions involved in the formation of carbamateduring the absorption of CO₂ by the erythrocyte, brought about a morerapid and more complete lysis of the erythrocytes than in the absence ofthese reagents.

The influence of the presence of a carbamate in a lysis reagent on thespeed of lysis is shown below in the experimental part.

That is why a subject of the present application is a multifunctionalreagent for erythrocytes characterized in that it contains a quantitysufficient to produce the lysis or the sphering of the erythrocytes sothat they can be detected and counted by a cytometer or an automaticcounting device,

Of a carbamate of a nitrogenated heterocycle or of a halide such asammonium chloride or

Of an agent inducing the formation by the erythrocytes, from carbonateand a nitrogenated heterocycle or ammonium ions, of a carbamate combinedwith the absorption of CO₂ by said erythrocytes.

The multifunctional reagent according to the invention permits eithereffective lysis of the erythrocytes or if desired the production oftheir simple sphering.

In preferential conditions for the implementation of the invention, thecarbamate can be used in the molar concentration of 0.000001M to 0.1M,particularly 0.00001M to 0.01M and quite particularly 0.0001M to 0.005M.In wholly preferential conditions for the implementation of the lysisreagent described above, a concentration of 0.0004M is used.

In other preferential conditions for the implementation of theinvention, the agent inducing the formation by the erythrocytes, fromcarbonate or an amine base, of a carbamate combined with the absorptionof CO₂ by said erythrocytes, is a catalyst agent of the reactionCO₂+H₂O

H₂CO₃such as carbonic anhydrase, for example carbonic anhydrase I or carbonicanhydrase II. The enzyme can have varied isoelectric points and can havevarious sources as their origin. The activity of the enzyme is expressedin Wilbur-Anderson units. The carbonic anhydrase can be present in theconcentration of 1 W-A U/liter to 1,000,000 W-A U/liter, particularlyfrom 10 W-A U/liter to 500,000 W-A U/liter and very particularly from100 W-A U/liter to 100,000 W-A U/liter. In wholly preferentialconditions for the implementation of the lysis reagent described above,a concentration of approximately 50,000 W-A U/liter is used.

In the present application and in the following, the nitrogenatedheterocycle in part constituting the carbamate can be for examplebicylic and preferably monocyclic. It can be unsaturated and in thiscase comprises for example 5, preferably 4, in particular 3,particularly 2 double bonds, and it is preferably saturated. Itcomprises for example from 3 to 8, in particular from 3 to 6 andparticularly from 3 to 5, and very particularly 4 or 5 carbon atoms. Itcomprises 2, in particular 1 single nitrogen atom.

As saturated nitrogenated heterocycle there can for example be mentionedpyrazolidine, imidazolidine, the imidazoline and piperazine, inparticular morpholine and particularly piperidine or pyrrolidine.

In other preferential conditions for the implementation of theinvention, the multifunctional reagent for erythrocytes moreovercontains a nitrogenated heterocycle or an ammonium salt such as a halidelike chloride.

The nitrogenated heterocycle can be one of those previously mentioned.

In a multifunctional reagent according to the invention, thenitrogenated heterocycle can be present in the molar concentration of0.01 to 0.250 M, particularly 0.08 to 0.19 M and very particularly 0.12to 0.18 M. In wholly preferential conditions for the implementation ofthe multifunctional reagent described above, a concentration of 0.17 Mis used.

The concentrations of the compound concerned in the reaction medium(multifunctional reagent+blood sample) during erythrocytic lysis arepreferably 0.01 to 0.225 M, particularly 0.072 to 0.17 M and veryparticularly 0.11 to 0.17 M.

In a multifunctional reagent according to the invention using carbonicanhydrase, the carbonate or hydrogen carbonate can be dispensed with asthe blood serum naturally contains them. In yet other preferentialconditions for the implementation of the invention, for example toaccelerate lysis, the multifunctional reagent for erythrocytes moreovercontains a carbonate or a hydrogen carbonate.

Sodium or potassium carbonate or hydrogen carbonate can for example bementioned.

In a multifunctional reagent according to the invention, the carbonateor the hydrogen carbonate can be present up to the molar concentrationof 0.1 M, particularly up to 0.01 M, and very particularly up to 0.005M. In very preferential conditions for the implementation of themultifunctional reagent described above, a concentration of 0.0025 M isused.

The concentrations of the compound concerned in the reaction medium(multifunctional reagent+blood sample) for erythrocytic lysis arepreferably 0.0001 to 0.1 M, particularly 0.001 to 0.01 M and veryparticularly 0001 to 0.005 M.

In yet other preferential conditions for the implementation of theinvention, the multifunctional reagent for erythrocytes moreovercontains a protective agent against the deterioration of the leucocytessuch as a fixation agent, in particular an aliphatic aldehyde such as inC₁-C₅, for example paraformaldehyde and particularly formaldehyde.

The aliphatic aldehyde can be present in a concentration of 0.01% to 5%,particularly 0.04% to 1% and very particularly 0.1% to 0.5%.

In yet more preferential conditions for the implementation of theinvention, the multifunctional reagent of the invention also comprisesan effective quantity of an anticoagulant agent.

Heparin, in particular ion citrate, EGTA and particularly EDTA can forexample be mentioned as anticoagulant agent.

The pH of the mixture without buffer at neutral pH during and afterlysis tends to increase, thus provoking cell degradation. The use of abuffer of approximately neutral pH is therefore desirable. Despite thefact that the buffer tends to inhibit lysis, a small quantity of buffer(for example 1-20 mM) can be used thanks to the accelerating effect of acatalyst such as carbonic anhydrase. The use of a carbamate according tothe invention allows the use of a buffer of approximately neutral pH.

This is why, in other preferential conditions for the implementation ofthe invention, the multifunctional reagent of the invention alsocomprises an effective quantity of a buffer agent particularly pH 6.5 to7.5. There can for example be mentioned as buffer agent MES(2-(N-morpholino)ethane sulphonic acid), in particular MOPS(3-(N-morpholino)propane sulphonic acid) and particularly HEPES(N-(2-hydroxyethyl)piperazine-N′-(2-ethane sulphonic acid)) andparticularly the buffer DIPSO pH 7.5.

In a multifunctional reagent according to the invention, the bufferagent can be present in the molar concentration of 0.0001 to 0.050 M,particularly 0.0005 to 0.03 M and very particularly 0.001 to 0.010 M.

In preferential conditions for the creation of the multifunctionalreagent described above without fixing agent, 0.17 M of pyrrolidinehydrochloride, 2.5 mM of potassium hydrogen carbonate, 3 mM of DIPSObuffer pH 7.5, 10 mg/l of carbonic anhydrase and 0.1 mM of EDTA areused.

In very preferential conditions for the creation of the multifunctionalreagent described above with fixing agent, 0.3% of formaldehyde is alsoused.

As seen above, the catalyst agent of the reactionCO₂+H₂O

H₂CO₃orcarbonate

carbamate+H₂Osuch as carbonic anhydrase, induces the formation by the erythrocytes,from carbonate or from an amine base, of a carbamate combined with theabsorption of CO₂ by said erythrocytes. The carbamates of thenitrogenated heterocycles can also be used directly to produce lysis ofthe erythrocytes. Some of these are new products.

This is why a subject of the present application is also a carbamatechosen from

pyrrolidine carbamate,

piperidine carbamate.

The reagents which are the subject of the present invention possess veryadvantageous properties. They are endowed in particular with remarkableproperties which are lytic with regard to erythrocytes.

These properties are illustrated below in the experimental part. Theyjustify the use of the reagents described above, in a method or aprocess for lysis or sphering of erythrocytes. Lysis of the red bloodcells allows the analysis and/or triage using a flow cytometer of theleucocytes and their sub-populations, platelets, red blood cell residuesafter lysis, or any other cellular or suspended, element added or not,such as for example spherules.

They also justify the use of the reagents described above, in a processfor the preparation of leucocytes.

A subject of the present application is also therefore a method of lysisof the erythrocytes in which a sample of whole blood treated with aanti-coagulant such as EDTA, EGTA, heparin or the citrate ions, issubjected to the action of a lysis reagent described above, to produceat least 95% lysis of the erythrocytes in less than 30 minutes.

It is possible to operate in the absence or in the presence ofmonoclonal antibodies in order to carry out the labelling of theleucocytic cells. These antibodies can be bound or not bound to afluorescent compound such as those described below. In preferentialconditions of implementation, these antibodies are bound to afluorescent compound.

The multifunctional reagents described above can be used as follows:

A sample of 0.1 ml of blood treated with an anti-coagulant, andincubated beforehand with a monoclonal antibody or a monoclonal antibodycombined with a fluorescence marker, or a mixture of monoclonalantibodies combined with fluorescence markers is brought into contactwith 2 ml of multifunctional reagent above and left for ten minutesduring which the lysis finishes. Such markers are for example CD45-FITC(CD45 combined with fluoresceine isothiocyanate) or CD14 combined withphycoerythrin and are marketed for example by the companies DAKO, BECTONand DICKINSON or BECKMAN COULTER. A reading in a cytometer for exampleof BECTON and DICKINSON Facscan or BECKMAN COULTER XL type is thencarried out either immediately, or up to 3 days after lysis.

In preferential conditions for the implementation of the method of lysisaccording to the invention, the preferential conditions described abovefor the multifunctional reagent are chosen.

The systems of lysis covered by the invention can also be used in thepresence of a permeation agent such as an aliphatic alcohol or adetergent. A permeation reagent allowing an intracellularimmunolabelling including a catalyst such as carbonic anhydrase or acarbamate is also part of the invention. Such a lysis and permeationstage can be carried out optionally after fixation by an aliphaticaldehyde.

In preferential conditions for the implementation of the processdescribed above, a sample of whole blood is brought into contact with aquantity of a reagent above sufficient to produce the lysis of at least95% of the erythrocytes in less than 10 minutes.

In other preferential conditions for the implementation of the processdescribed above, the pH of the mixture of the reagent and the sample isbetween 4 and 9.

But the multifunctional reagent according to the invention also allowsthe production of the simple sphering of the erythrocytes.

This is why a subject of the present application is also a process ofsphering (swelling) of the erythrocytes characterized in that a sampleof whole blood treated with an anticoagulant is brought into contactwith a quantity of a reagent above sufficient to provoke the sphering ofthe erythrocytes in such a way as to allow their analysis by anautomatic counting device.

In preferential conditions for the sphering of the erythrocytes a sampleof blood is brought into contact with a quantity of sphering reagentsufficient to produce sphering in 10 second to 400 second intervals. Toavoid lysis of the erythrocytes in a time interval and to control thespeed of the sphering, the multifunctional reagent according to theinvention can be diluted in a preferably isotonic solution. Between 0and 10 times the same volume of diluent, preferably between 0.5 and 5times and particularly between 1 and 3 times can for example be added.In particularly preferential conditions, the choice of isotonic solutionis IsoFlow® from Beckman Coulter which contains NaCl: 7.92 g/l, KCl: 0.4g/l, NaH₂PO₄: 0.19 g/l, Na₂HPO₄: 1.95 g/l, EDTA: 0.38 g/l,2-phenoxyethanol: 3 g/l, and NaF: 0.3 g/l.

In other preferential conditions, one or more adjuvants such as fixationreagents, colorants of nucleic acids, and/or isosphering agents such asmaltoside (D. H. Tycko, U.S. Pat. No. 5,194,909) are added to thesphering reagent.

As has been seen above, the multifunctional reagent of the invention isnot aggressive for the leucocytes and can therefore be used with a viewto their preparation. The lysis systems covered by the invention aretherefore also tools for the preparation of viable leucocytic cells andcan replace the technique of cell separation on Ficoll-Hypaque. Thepreparation of the viable cells after lysis of blood, pathologicalblood, bone marrow and any other body fluid, has an application forexample for the purposes of research for functional cellular tests,freezing and storage of the cells, etc. As far as the control ofcellular viability is concerned, the lysis methods can be used withviability colorants such as eosin, blue tryptan, 7-AAD, LDS 751 etc.Finally, as the lysis according to the studies of the Applicant is afunction of the capacity of the erythrocytes to absorb CO₂, the systemof lysis can be used as a functional parameter of the erythrocytes.

This is why a subject of the present application is also a process forthe preparation of leucocytes characterized in that it comprises a stageof lysis of the erythrocytes using a multifunctional reagent such asdescribed above.

The preferential conditions for the implementation of the processesdescribed above also apply to the other subjects of the inventionreferred to above.

FIG. 1 represents a size-structure diffusion diagram of a blood sampleanalysed using flow cytometry (BECKMAN COULTER XL) after lysis accordingto Example 5. Region A corresponds to the lymphocytes, region Bcorresponds to the monocytes and region C to the granulocytes.

FIGS. 2A-2E represent size-structure diffusion diagrams of a bloodsample analysed using flow cytometry (BECKMAN COULTER XL), aftersphering according to Example 9. After contact of the erythrocytes withthe sphering agent, described in Example 6, the size-structureoccurrences of the erythrocytes are represented in 4 intervals of 100seconds (FIG. 2A). The sphering and the homogenization of theerythrocytes which results from this over time are illustrated in thesize-structure diagrams; FIG. 2B, after 100 seconds; FIG. 2C, after 200seconds; FIG. 2D, after 300 seconds; FIG. 2E, after 400 seconds.

The following examples illustrate the present application.

EXAMPLE 1

A multifunctional reagent for erythrocytes having the followingcomposition was prepared: Pyrrolidine chloride 170 mM Potassium hydrogencarbonate 2.5 mM DIPSO buffer pH 7.5 3 mM Bovine carbonic anhydrase(SIGMA) 10 mg/l (50000 W-A units) EDTA 0.1 mM pH 7.3

EXAMPLE 2

A multifunctional reagent for erythrocytes having the followingcomposition was prepared: Pyrrolidine chloride 170 mM Potassium hydrogencarbonate 2.5 mM DIPSO buffer pH 7.5 3 mM Bovine carbonic anhydrase(SIGMA) 10 mg/l (50000 W-A units) EDTA 0.1 mM Formaldehyde 0.1% pH 7.3.

EXAMPLE 3

A multifunctional reagent for erythrocytes having the followingcomposition is prepared: Piperidine chloride 170 mM Potassium hydrogencarbonate 2.5 mM DIPSO buffer pH 7.5 3 mM Bovine carbonic anhydrase(SIGMA) 10 mg/l (50000 W-A units) EDTA 0.1 mM pH 7.3.

EXAMPLE 4

A multifunctional reagent for erythrocytes having the followingcomposition was prepared: Ammonium chloride 155 mM Potassium hydrogencarbonate 2.5 mM DIPSO buffer pH 7.5 3 mM Bovine carbonic anhydrase(SIGMA) 10 mg/l (50000 W-A units) EDTA 0.1 mM pH 7.3

EXAMPLE 5 Performance of a Lysis

100 μl of whole blood is mixed with 1 ml of a lysis agent of Example 1.The solution is left to rest at ambient temperature for 10 minutes andthe leucocytes are counted by cytometry.

EXAMPLES 6 TO 8 Preparation of Pyrrolidine Piperidine and MorpholineCarbamates

A mixture of sodium bicarbonate and a quantity of each compound (molarratio 1:2), was heated to 100.degree. C. accompanied by stirring. Thereaction is characterized by a solidification of the mixture, whichoccurs after approximately an hour. The sought carbamates were recoveredafter extraction with methanol. The molecular structure of thesesynthesized compounds was verified by NMR spectrometry ¹³C at 200 MHz.

These analyses (in deuterated methanol) made it possible to obtain thefollowing chemical shifts (δ: ppm):

1/Pyrrolidine carbamate: 26.44; 27.24; 47.32; 47.63 (CH₂; 165.02 (C═O)

2/Piperidine carbamate: 25.69; 26.58; 27.39; 28.71; 47.65 (CH₂; 162.76(C═O)

3/Morpholine carbamate: 47.14; 68.42 (CH₂; 162.81 (C═O)

EXAMPLE 9 Preparation of a Sphering Agent

A sphering agent was prepared by mixing 2 volumes of reagent accordingto Example 1 and a volume of Isoflow®, an isotonic composition marketedby BECKMAN COULTER.

EXAMPLE 10 Cytometry After Sphering

A volume of blood of 4 microliters was placed in contact with 1 ml ofthe sphering agent of Example 9. The process of sphering was tracked bycytometry as a function of time in a BECKMAN COULTER XL cytometer. Theresults are presented in FIGS. 2A-2E mentioned above.

EXPERIMENT 1

The speed of lysis by the ammonium ion and by nitrogenated heterocyclesin the presence of KHCO₃ (10 mM) or in the presence of their respectivecarbamate derivatives (0.4 mM) was studied. A volume of 2 ml of reagentwas used for a volume of 0.1 ml of whole blood treated withanticoagulant (EDTA). Variations in pH were obtained by adding a 1 Maqueous solution of HCl before the addition of carbamate. The carbamates(100 mM in methanol) were added to the mixtures at the start of thereaction. The pH of the reaction mixture was measured. The lysis processwas tracked spectrophotometrically (measurement of optical density at700 nm). The lysis time is determined by the time necessary to obtain aminimum level.

The results obtained are the following: Time necessary pH during toreach total lysis the lysis (maximum transparency) reaction NH₄Cl (150mM), 7 mn 7.40 KHCO₃ (10 mM) Idem 4 mn 7.03 NH₄Cl (150 mM), 1 mn 50 7.20ammonium carbamate (0.4 mM) Pyrrolidine (150 mM), 7 mn 7.20 KHCO₃ (10mM) Idem 6 mn 7.75 Pyrrolidine, pyrrolidine 3 mn 7.66 carbamate (0.4 mM)Piperidine (150 mM), 15 mn 7.30 KHCO₃ Idem 10 mn 7.8 Piperidine (150mM), 6 mn 40 7.68 piperidine carbamate (0.4 mM) Morpholine (150 mM), 12mn 6.82 KHCO₃ (10 mM) Morpholine (150 mM), 8 mn 6.90 Morpholinecarbamate (0.4 mM)

Conclusion: It will be seen that the addition of the differentcarbamates to the ammonium ion and to the nitrogenated heterocyclesinduces a much greater speed of lysis than that obtained by addinghydrogen carbonate at a concentration 25 times that of the carbamate. Asthe table shows, the lytic effect of the carbamates does not depend onthe pH.

EXPERIMENT 2 Demonstration of the Catalyst Role of Carbonic Anhydrase inthe Reaction CarbonateCarbamate+H₂O

The reaction (NH₄)₂CO₃

NH₂CO₂NH₄+H₂O was studied through the conversion of the ammoniumcarbamate into ammonium carbonate measured by their difference insolubility in an aqueous solution of 85% of acetone in which thecarbonate precipitates and not the carbamate. Duration before completeprecipitation in Reaction mixture 85% of acetone 100 mM NH₂CO₂NH₄ 240min 100 mM NH₂CO₂NH₄ + 0.001 mg/ml of  10 min carbonic anhydrase

Conclusion: The results show that the carbonic anhydrase, in addition tocatalysing the conversion CO₂+H₂O

H₂CO₃ as is well known, also catalyses the conversion carbonate

carbamate+H₂O

EXPERIMENT 3 Demonstration of the Effect on the Speed of Lysis ofCarbonic Anhydrases Originating from Different Sources

In a lysis reagent according to Example 1, the component carbonicanhydrase at 50,000 W-A U/I was replaced by preparations of carbonicanhydrase as indicated in the table, each at 5000 W-A U/I. The lysisprocess was tracked spectrophotometrically (measurement of the OD at 700nm). The duration of the lysis is determined by the time necessary toachieve a minimum level. Speed of lysis at Supplier Reference Origin5000 W-A U/l Control without enzyme 15.00 min  Sigma C-3934 Bovine 7.30min Biozyme CABI Bovine 7.06 min Biozyme CABII Bovine 8.30 min SigmaCAII, C-2522 Bovine 8.51 min Sigma CAII, C-6165 Human 7.06 min SigmaCAI, C-4396 Human 5.06 min Sigma CAI, C-5290 Human 3.24 min Sigma CAI,C-1266 Rabbit 8.00 min

Conclusion. The different preparations of carbonic anhydrase allincrease the speed of lysis.

1. A method of lysis of erythrocytes comprising: (a) providing a lysisreagent comprising a carbonic anhydrase and an ammonium salt or aheterocyclic amine or salt thereof; and (b) mixing a blood sample withsaid lysis reagent to lyse erythrocytes in formed mixture.
 2. The methodof claim 1 further comprising analysis of leukocytes in said mixture ona flow cytometer.
 3. The method of claim 2 further comprising analysisof subpopulations of said leukocytes in said mixture on said flowcytometer.
 4. The method of claim 2 further comprising incubating saidblood sample with an antibody or a fluorescence marker to labelleukocytes, prior to mixing said blood sample with said lysis reagent.5. The method of claim 4 further comprising analysis of labeledleukocytes on said flow cytometer.
 6. The method of claim 1, whereinsaid heterocyclic amine comprises pyrazolidine, imidazolidine,imidazoline, piperazine, morpholine, piperidine, or pyrrolidine.
 7. Themethod of claim 1, wherein said lysis reagent further comprises acarbonate or hydrogen carbonate salt.
 8. The method of claim 7, whereinsaid method produces lysis of at least 95% of said erythrocytes in saidmixture in less than 30 minutes.
 9. The method of claim 8 furthercomprising analysis of leukocytes in said mixture on a flow cytometer.10. The method of claim 7 further comprising incubating said bloodsample with an antibody or a fluorescence marker to label leukocytes,prior to mixing said blood sample with said lysis reagent.
 11. Themethod of claim 10 further comprising analysis of labeled leukocytes onsaid flow cytometer.
 12. A method of lysis of erythrocytes comprising:(a) providing a lysis reagent comprising an ammonium salt or aheterocyclic amine or salt thereof and a catalyst agent enablinginducing formation of carbamate in the presence of erythrocytes; and (b)mixing a blood sample with said lysis reagent and forming a mixture,wherein said catalyst agent induces formation of carbamate in saidmixture, thereby causing lysis of erythrocytes in said mixture.
 13. Themethod of claim 12, wherein said catalyst agent is a carbonic anhydrase.14. The method of claim 13 further comprising analysis of leukocytes insaid mixture on a flow cytometer.
 15. The method of claim 14 furthercomprising analysis of subpopulations of said leukocytes in said mixtureon said flow cytometer.
 16. The method of claim 13 further comprisingincubating said blood sample with an antibody or a fluorescence markerto label leukocytes, prior to mixing said blood sample with said lysisreagent.
 17. The method of claim 16 further comprising analysis oflabeled leukocytes on said flow cytometer.
 18. The method of claim 13,wherein said lysis reagent further comprises a carbonate or hydrogencarbonate salt.
 19. The method of claim 18, wherein said method produceslysis of at least 95% of said erythrocytes in said mixture in less than30 minutes.
 20. The method of claim 18 further comprising analysis ofleukocytes in said mixture on said flow cytometer.
 21. The method ofclaim 18 further comprising incubating said blood sample with anantibody or a fluorescence marker to label leukocytes, prior to mixingsaid blood sample with said lysis reagent.
 22. The method of claim 21further comprising analysis of labeled leukocytes on said flowcytometer.
 23. The method of claim 12, wherein said heterocyclic aminecomprises pyrazolidine, imidazolidine, imidazoline, piperazine,morpholine, piperidine, or pyrrolidine.
 24. A method of spheringerythrocytes comprising: (a) mixing a blood sample with an isotonicdiluent and a lysis reagent comprising a carbonic anhydrase and anammonium salt or a heterocyclic amine or salt thereof to sphereerythrocytes; and (b) analyzing sphered erythrocytes on a flowcytometer.
 25. The method of claim 24, wherein said lysis reagentfurther comprises a carbonate or hydrogen carbonate salt.